1. Field of the Invention
This invention relates to the formation of protein crystals and more particularly to a method and apparatus for dynamically controlling the process of forming protein crystals as well as to the crystals formed by the method and apparatus.
2. Description of the Related Art
The concept of rational drug design involves obtaining the precise three dimensional molecular structure of a specific protein to permit design of drugs that selectively interact with and adjust the function of that protein. Theoretically, if the structure of a protein having a specified function is known, the function of the protein can be adjusted as desired. This permits a number of diseases and symptoms to be controlled. For example, CAPTOPRIL is a well known drug for controlling hypertension that was developed through rational drug design techniques, CAPTOPRIL inhibits generation of the angiotension-converting enzyme thereby preventing the constriction of blood vessels. The potential for controlling disease through drugs developed by rational drug design is tremendous.
X-ray crystallography techniques are utilized to obtain a "fingerprint", i.e. the precise three-dimensional shape, of a protein crystal. However, a critical step to rational drug design is the ability to reliably crystallize a wide variety of proteins. Therefore, a great deal of time and money have been spent crystallizing proteins for analysis. Recent biotechnological developments in protein cloning, over expression, and affinity purification of proteins will likely increase the need for a reliable way in which to grow various protein crystals. Unfortunately protein crystallization is quite difficult.
Protein crystallization involves the creation of a supersaturated protein solution under conditions that promote minimum protein solubility and the orderly transition of macromolecules from the solution into a crystal lattice. The variables that must be controlled precisely to promote crystal growth include temperature, protein solution concentration, salt solution concentration and pH, for example. These variables are carefully controlled and optimum combinations thereof are determined through experimentation to yield superior crystals.
The crystallization process generally involves three distinct phases; nucleation, sustained crystal growth, and termination of crystal growth. Nucleation is the initial formation of an ordered grouping of a few protein molecules and requires a protein in a salt solution at a particular concentration. On the other hand, the continued growth phase consists of the addition of protein molecules to the growing faces of the crystal lattice and requires lower concentrations of salt solution than the nucleation phase. The termination phase can be initiated by poisoning the growing lattice with denatured protein or a different protein, by depletion of the protein solution or by changing the salt concentration to a specified level. The protein is often referred to as a "reactant" and the salt solution is often referred to as a "reagent" in crystallization processes.
It is considered desirable to obtain a small number of crystallization nuclei quickly that will grow slowly into full size crystals. Theoretically, this allows for a relatively large size of the resulting crystals, homogenous crystal order and morphology and balanced crystal dimensions. Therefore, it is desirable to begin crystallization with a particular salt concentration until nucleation is detected, at which point it is desirable to adjust the concentration of salt. Thus, one of the critical requirements of any protein crystallization process is the fine and dynamic control of the various parameters that determine the concentration of the salt solution in which the target protein is suspended. This control requires the ability to attain nucleation conditions and the ability to modify the concentration of the salt solution without disturbing the crystallization process.
There are several conventional techniques for forming protein crystals; for example, liquid diffusion, vapor diffusion and dialysis techniques. These processes are relatively slow and cannot readily be controlled dynamically. Therefore, these processes require complex and largo apparatus in order to control crystallization if crystallization can be controlled at all, Accordingly, it is desirable to overcome these limitations.
Most conventional crystallization methods mix a protein solution with a crystallizing (or precipitant) solution to accomplish crystallization. In terms of mechanics, use is commonly made of syringes, stepping motors, valves of various types, membranes to separate solutions, and in one case, a gel to replace the membrane and act as a delaying filter device between solutions. U.S. Pat. Nos. 4,917,707, 5,106,592, 5,641,681 and Microdialysis Crystallization Chamber, L. C. Sieker, J. Crystal Growth 90 (1988) 349-357, the entire contents of which are incorporated herein by reference, disclose these concepts.
It is known to "control" the crystallization process. However, only the movement of liquids via pumps, valves and syringes is controlled in conventional crystallization processes. This control creates a static condition (bath concentration) which is predefined for the protein in question. For example, U.S. Pat. No. 4,755,363, the entire contents of which are incorporated herein by reference, discloses delivering liquids at desired flow rates and concentrations, However, U.S. Pat. No. 4,755,363 fails to disclose changing conditions within the crystallization chamber (with the exception of temperature) once those conditions have been set and crystallization has begun.
Temperature is an important parameter that can be controlled to optimize conditions separately for nucleation or growth. U.S. Pat. Nos. 4,755,363 and 5,362,325, the entire contents of which are incorporated herein by reference, are exemplary of patents disclosing temperature control in crystallization processes. U.S. Pat. No. 5,362,325 discloses varying the concentration of a crystallizing agent, over time, to produce a predetermined gradient in the concentration of the crystallizing agent. However, this reference fails to disclose dynamic control.
Automation is a recent trend in crystallography. Robotics enables systematic pipetting of solutions and protein into crystal growth chambers on plates, so that a multiplicity of conditions can be examined more quickly and consistently. The use of robotics frees valuable time for researchers. Another trend is the us- of semi-automated techniques to record results.
Also, an inherent limitation in any crystallization process is the effects of molecular convection, thermal effects and buoyancy, all due to the earth's gravitational field. Therefore, crystallization experiments have been proposed and conducted in microgravity (1/1000 g to 1/10,000 g) on board the Space Shuttle, International Space Station, and other vehicles. Several patents disclose crystallization in microgravity to improve the size, morphology and diffraction quality of crystals. U.S. Pat. Nos. 5,362,325 and 4,755,363, which are incorporated herein by reference, are exemplary of patents disclosing microgravity crystallization. In fact, a low gravity environment is "crucial" (emphasis added) to the success of one apparatus (Sygusch, et al. J. Crystal Growth 162 (1996) 167-172). Early experiments indicate that larger and more homogenous crystals can be grown in microgravity environments by eliminating the effects of the earth's gravitational field. However, the practical limitations of using current space vehicles, such as the Space Shuttle, render it difficult to use conventional apparatus/methods for crystallization in microgravity environments. Particularly, conventional apparatus are too large, are difficult to control remotely and automatically, have many moving parts that can fail, do not permit accurate change of solution concentration during the process and are not entirely reusable. Also, known processes require too much time to produce fully grown crystals. Therefore, it is desirable to overcome these deficiencies to permit crystal growth experiments in space under microgravity conditions. Also, conventional apparatus and methods do not facilitate experimentation in which the only variable is the presence or absence of the earth's gravitational field because conventional crystallization apparatus must be modified significantly for use in space. Moreover, the foregoing methods and apparatus do not provide a dynamic control capability in either the earth's gravitational field or in microgravity.